Supporting Information

for

Bioaccumulation behavior of pharmaceuticals and personal care products in adult

zebrafish (Danio Rerio): influence of physical-chemical properties and biotransformation

Fangfang Chen1, Zhiyuan Gong1, 2, Barry C. Kelly3*

1
Graduate School of Integrated Sciences and Engineering (NGS), National University of Singapore,

Singapore 117456, Singapore

2
Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore
3
Department of Civil and Environmental Engineering, National University of Singapore,

Singapore 117576, Singapore

* Corresponding author. Present address: Department of Civil and Environmental Engineering,

National University of Singapore, Block E1A, #07-03, No.1 Engineering Drive 2, Singapore
117576. Tel. (+65-6516 3764); Fax (+65-6779 1635); Email: [email protected]

This PDF file includes:

Standards and Reagents

Experimental setup and collection of samples
Data analysis
Estimation of size and temperature corrected biotransformation rate constant (km) values
Estimation of bovine serum albumin / muscle protein-water distribution coefficient
Supplemental Tables S1-S8
Supplemental Figure S1-S5
Supplemental References
Standards and Reagents. Labeled surrogate standards including propanolol-d7, ibuprofen-d3,
fluoxetine-HCl-d5, carbamazepine-d10, warfarin-d5 were purchased from CDN Isotopes Inc.
(Pointe-Claire, Quebec, Canada). Bisphenol A-d6, 2,4,5- trichlorophenoxyacetic acid (TCPAA)-
13
C6 and gemfibrozil-d6 were purchased from Cambridge Isotope Laboratories, Inc. (Andover,
MA, US). Triclosan-13C12 was purchased from Wellington Laboratories Inc. (Ontario, Canada).
All the standards were prepared in methanol and stored at -20oC. HPLC grade Acetone, methanol
and acetonitrile were purchased from Fisher Scientific Inc. (Loughborough, UK).
Experimental setup and collection of samples. Adult female zebrafish was maintained in
dechlorinated water at a temperature of 25 ± 2°C with a 16: 8 hour light/dark cycle. The
bioconcentration experiment consisted of two phases, namely the uptake and depuration phase.
The uptake phase was continuous aqueous exposure of a mixture of multiple pharmaceuticals
and personal care products (PPCPs) over a 6-d period, followed by a 7-d depuration phase with
clean water in fish tanks. 280 randomly selected fish were exposed to the mixture of PPCPs in
low dose (L) group and another 280 fish in a high dose (H) group. A separate group of 280 fish
kept in clean water served as the control group. The low dose group was at concentrations
similar to environmental levels. All flow-through experiments started at least two days before the
introduction of the fish in order to allow the exposure tanks to equilibrate with the target test
concentration. Artemia were fed to fish twice daily during the exposures.
Data analysis. Observed concentrations in fish tissues were corrected by growth dilution. The
whole-body growth rate is being used to correct concentrations for each tissue. This assumes that
the growth rates for the tissues are all the same and proportional to the whole body. The
exponential growth model was utilized to predict the fish weight (FW), 1

FW = a·exp(g·t) (S1)

where a is a constant, g is the growth rate, and t is time. Equation S1 was used to determine the
increase in FW at each sampling interval, relative to t = 0 in order to correct measured
concentrations in zebrafish samples for growth dilution. The depuration rate constant (kd) was
determined using equation S2 by fitting the growth corrected concentrations (Cfish(t)) during the
depuration phase to the first-order decay model,

Cfish(t) = a·exp(-kd·t) (S2)

where a is a constant, and t is time. The half-life was calculated as ln(2)/kd.

Iterative nonlinear regression (Origin, Version 9.0, Northhampton, MA, USA) was used to
determine uptake rate constants (ku),

C fish(t) = [(ku )(Cw )/(kd )]·[1- exp(-kd ·t)] (S3)

where Cw is the average water concentration and kd is the depuration rate constant. 1, 2
Kinetically-derived bioconcentration factors (BCFk) were calculated by ku/kd. In addition, a
steady- state bioconcentration factor (BCFss) was determined using the growth-corrected

S1
concentration in zebrafish at the end of the uptake phase divided by the constant concentration in
water (i.e., BCFss = Cf(ss)/Cw).

Estimation of size and temperature corrected biotransformation rate constant (km) values.
Biotransformation rate constant (km) values compiled from EPI Suite BCFWIN were corrected to
represent estimated values for a 1 g fish at 25 °C, which accurately reflects the conditions of the
present study. The equation used to convert these quantitative structure activity relationship
(QSAR) derived km values is provided in the EPI Suite BCFWIN software module and is shown
below:

km,X = km,N (WX/WN)-0.25 exp(0.01(TX-TN))

where km,X is the temperature and size corrected biotranformation rate constant (km) value of the
test chemical, km,N is the original QSAR estimated value of km for a 10 g fish at 15 °C, TN is the
temperature in the original QSAR model (i.e., 15 °C), TX is the temperature of the actual
conditions (in our case 25 °C), WN is the weight of the fish in the original QSAR model (10 g
fish) and WN is the weight of the desired fish species (1 g for zebrafish).
Estimation of bovine serum albumin / muscle protein-water distribution coefficient. Bovine
serum albumin-water distribution coefficient, DBSAw (pH 7.4) values were estimated following
the approach outlined by Henneberger et al. 3 for ionizable organic chemicals (IOCs) using
polyparameter linear free energy relationship (PP-LFER) models. Experimentally determined
descriptors for neutral species (E, S, A, B, V) was taken from the UFZ-LSER database. 4 Where,
E is the excess molar refraction, S is the polarizability/dipolarity parameter, A represents the H-
bond donor properties and B the H-bond acceptor properties, and V is the molar volume. The
model predicted descriptors were from the ABSOLV module in the ACD/Percepta software
(2015 release). The descriptors of the neutral species were then utilized to calculate descriptors
for the ionic species (Ei, Si, Ai, Bi, Vi, Ji+, Ji-) using the empirical equations from Abraham et al. 5,
6
Equations are only available for phenols, carboxylic acids, pyridine, and amines. Subsequently,
DBSAw (pH 7.4) values were estimated using following equation,
Log DBSAw [L/kg] = 0.85(0.19) + 0.63(0.08)!" − 0.63(0.07)#" − 0.05(0.22)$" −2.08(0.19)%" +
2.06(0.20)&" − 1.16(0.27)'"+ + 3.13(0.28)'"- (S4)

Similarly, the muscle protein-water distribution coefficient, Dmpw (pH 7) values were estimated
using PP-LFER models as described by Henneberger et al. 7 for IOCs,

Log Dmpw [L/kg, dry weight] = -0.237(0.157) + 0.675(0.067)!" − 0.764(0.083)#" −

0.196(0.146)$" −2.285(0.120)%" + 2.511(0.143)&" – 0.682(0.188)'"+ + 2.89(0.209)'"- (S5)

S2
Table S1. MRM transitions and MS parameters for target PPCP compounds and isotope labeled surrogate standards.
Retention Entrance Collision
Ion Transitions Dwell time Declustering Collision cell exit
Compound Time Polarity Potential energy
(m/z)a (ms) Potential (V) potential (V)
(min) (V) (eV)
Naproxen 229.2 > 169.2 2.51 20 - -50 -6 -41 -6
229.2 > 170 2.51 20 - -50 -6 -19 -9
Warfarin-d5 312.1 > 160.9 2.59 20 - -236 -12 -34 -9
TCPAA-13C6 b 259 > 201 2.76 20 - -34 -10 -19 -7
Ibuprofen 205 > 159 3.39 20 - -37 -3 -9 -5
Ibuprofen-d3 208.2 > 163.8 3.37 20 - -45 -7 -12 -12
Diclofenac 294 > 250 3.39 20 - -34 -10 -15 -5
294 > 34.9 3.39 20 - -34 -10 -54 -4
Carbamazepine 237.1 > 194 3.61 20 + 187 7 27 11
Carbamazepine-d10 247.1 > 204.1 3.61 20 + 156 9 27 12
Bisphenol A 227.1 > 133 3.86 20 - -130 -7 -31 -8
Bisphenol A-d6 233.1 > 138 3.86 20 - -120 -11 -33 -7
Gemfibrozil 249. 1> 121 4.08 20 - -60 -4 -20 -6
Gemfibrozil-d6 255.3 > 121.1 4.08 20 - -258 -10 -22 -6
Risperidone 411.5 > 190.8 4.27 20 + 238 5 28 14
411.5 > 110 4.27 20 + 238 5 69 7
Proponolol-d7 267.2 > 116 4.34 20 + 209 11 25 6
Diphenhydramine 256.1 > 167.1 4.65 20 + 100 11 16 10
256.1 > 165.1 4.65 20 + 100 11 58 10
Triclosan 286.8 > 35 4.84 20 - -80 -11 -39 -9
Triclosan-13C12 299 > 35 4.84 20 - -57 -10 -39 -6
Norfluoxetine 296.1>134.1 5.01 20 + 41 3 10 23
Fluoxetine 310.2 > 44.1 5.11 20 + 61 8 54 11
Fluoxetine-d5 315.4 > 44 5.11 20 + 151 7 57 11
Simvastatin 399 > 115 5.16 20 - -55 -3 -31 -2
Sertraline 306.2 > 159 5.37 20 + 45 5 36 9
306.2 > 275.2 5.37 20 + 45 5 17 51

S3
a
For compounds with two ion transitions, the first MRM transition was used for quantification and the second for confirmation. Only one MRM transition was
monitored for internal standards.
b 13
C6-2,4,5-trichlorophenoxyacetic acid used as injection internal standard for determining recovery of internal surrogate compounds spiked.

S4
Table S2. Results of spike-recovery experiments (Relative Recovery, %) for individual PPCPs in zebrafish plasma, tissues and
exposure water.

Compound Recovery (%) ± SD

Liver Ovary Muscle Plasma Water
Naproxen 44.8 ± 11 146 ± 10.8 173 ± 20 126 ± 26.0 53.8 ± 3.6
Ibuprofen 104 ± 3.1 117 ± 14.9 55.3 ± 8.2 97.6 ± 12.9 61.3 ± 16.3
Diclofenac 110 ± 6.3 79.5 ± 26.6 54.9 ± 10.1 108 ± 18.3 62.7 ± 16.1
Gemfibrozil 109 ± 6.7 166 ± 40.3 105 ± 17.5 100 ± 17.1 45.7 ± 11.5
Risperidone 89.2 ± 3.5 153 ± 16.7 78.8 ± 7.4 105 ± 3.7 98.4 ± 26.4
Diphenhydramine 76.7 ± 1.9 118 ± 26.5 118 ± 17.5 54.5 ± 5.3 86.9 ± 16.4
Norfluoxetine 72.7 ± 13.6 124 ± 4.4 147 ± 20.5 64.2 ± 1.9 75.8 ± 15.8
Fluoxetine 68.2 ± 3.7 161 ± 16.7 145 ± 10.7 74.3 ± 4.7 76.6 ± 12.8
Sertraline 54.2 ± 1.9 73.8 ± 33.4 88.4 ± 10.5 90.6 ± 5.2 77.8 ± 16.0
Carbamazepine 182 ± 10.5 141 ± 2.4 115 ± 8.8 93.4 ± 9.1 98.8 ± 13.4
Bisphenol A 65.9 ± 3.0 138 ± 7.9 136 ± 7.7 113 ± 8.5 88.2 ± 6.1
Simvastatin 76.1 ± 4.4 200 ± 54.2 203 ± 35.2 144 ± 16.2 137 ± 12.6
Triclosan 86.2 ± 4.7 180 ± 30.6 173 ± 26.3 99.4 ± 2.8 89.9 ± 9.3

S5
Table S3. Method detection limits (MDLs) and method quantification limits (MQLs) in fish tissues, plasma, as well as exposure water.

Compound Liver Ovary Muscle Plasma Water

MDL MQL MDL MQL MDL MQL MDL MQL MDL MQL
(ng×g-1 wet (ng×g-1 (ng×g-1 (ng×g-1 (ng×g-1 (ng×g-1 (pg×µL-1) (pg×µL-1) (pg×µL-1) (pg×µL-1)
weight) wet wet wet wet wet
weight) weight) weight) weight) weight)
Naproxen 0.336 1.12 0.008 0.027 0.009 0.030 0.352 1.17 0.052 0.173
Ibuprofen 0.085 0.283 0.384 1.28 0.031 0.103 0.226 0.755 0.013 0.042
Diclofenac 0.274 0.913 0.099 0.330 0.035 0.117 0.478 1.60 0.012 0.040
Gemfibrozil 0.227 0.757 0.026 0.087 0.016 0.053 0.088 0.295 0.062 0.208
Risperidone 0.002 0.007 0.001 0.003 0.0005 0.002 0.015 0.050 0.0002 0.001
Diphenhydra
mine 1.11 3.68 0.012 0.040 0.044 0.147 0.075 0.249 0.003 0.012
Norfluoxetine 0.424 1.41 0.051 0.171 0.057 0.189 0.985 3.285 0.018 0.061
Fluoxetine 0.302 1.01 0.041 0.137 0.069 0.230 1.30 4.33 0.0004 0.001
Sertraline 0.023 0.077 0.009 0.030 0.063 0.210 0.421 1.40 0.0003 0.001
Carbamazepi
ne 0.238 0.793 0.018 0.060 0.015 0.050 0.179 0.596 0.016 0.052
Bisphenol A 12.1 40.2 2.81 9.37 1.65 5.50 8.11 27.0 0.025 0.084
Simvastatin 0.929 3.10 0.509 1.70 0.072 0.240 0.115 0.382 0.011 0.036
Triclosan 0.171 0.570 0.729 2.43 0.125 0.417 0.394 1.31 0.022 0.074

S6
 Table S4. Calculated BSA-water distribution coefficient (log DBSAw, L×kg-1 pH 7.4), muscle protein-water distribution coefficient
(log Dmpw, L×kg-1 dry weight pH 7), and substance descriptors of test chemicals. Experimental descriptors were taken from the UFZ-
LSER database.4 The pKa value is only required for phenols for the calculation of the PP-LFER descriptors of ionic species.

log log Source of

DBSAw Dmpw descriptors E S A B V Ei Si Ai Bi Vi j i+ j i- Nai pKa
Carboxylic acids
Ibuprofen 3.37 1.35 Database 0.73 0.70 0.56 0.79 1.78 0.88 3.27 0.08 3.53 1.76 0.00 2.47
Diclofenac 4.12 1.99 Database 1.81 1.85 0.55 0.77 2.03 1.96 5.31 0.03 3.35 2.00 0.00 2.62
Gemfibrozil 3.66 1.70 Absolv 0.81 1.41 0.55 0.80 2.12 0.96 4.09 0.07 3.50 2.10 0.00 2.48
Naproxen 3.45 1.32 Database 1.51 1.98 0.60 0.68 1.78 1.66 5.04 0.02 3.13 1.76 0.00 2.43
Amines
Diphenhydramine 0.97 1.27 Database 1.31 1.11 0.00 1.22 2.19 1.16 3.94 1.46 0.00 2.21 2.24 0.00 1
Fluoxetine 2.37 2.18 Database 1.00 1.30 0.10 0.93 2.24 0.85 3.33 2.18 0.00 2.26 1.26 0.00 2
Norfluoxetine 1.64 1.44 Absolv 1.23 1.27 0.17 1.11 2.10 1.08 3.75 2.38 0.00 2.12 1.53 0.00 3
Sertraline 2.42 2.56 Absolv 1.83 1.50 0.10 0.82 2.27 1.68 3.16 2.18 0.00 2.29 1.81 0.00 2
Phenol
Triclosan 7.09 4.29 Database 1.85 1.69 0.74 0.29 1.81 2.00 7.77 0.00 3.01 1.79 0.00 3.98 7.8
Neutral compounds
Bisphenol A 2.77 2.05
Database
1.61
1.56
0.99
0.91
1.86 1.61 1.56 0.99 0.91 1.86 0.00 0.00
Carbamazepine 2.32 1.58 Database 2.15 1.90 0.50 1.15 1.81 2.15 1.90 0.50 1.15 1.81 0.00 0.00
Simvastatin 3.27 3.02 Absolv 1.35 2.55 0.32 1.86 3.427 1.35 2.55 0.32 1.86 3.427 0.00 0.00
Other
Risperidone NAa NAa
a
The model is not available for this compound.

S7
 Table S5. Uptake and depuration phase duration (d), fish mass (g), growth rate (g/d) and the associated coefficient of determination
(r2), mortality (%), and liver somatic index (%) for exposed and control zebrafish used in bioconcentration experiments.

Uptake Depuration Mean initial Liver somatic

period (d) period (d) fish mass (g) Growth rate (g/d) (r2) Mortality (%) index (%)
Control 6 7 0.82 0.0188 (0.86) 0 2.75
Low Group 6 7 0.81 0.0208 (0.96) (p = 0.46) 0 2.76 (p = 0.52)
High Group 6 7 0.84 0.0158 (0.92) (p = 0.48) 0.56 3.25 (p = 0.83)

S8
Table S6. Rate of uptake (ku ± standard error), rate of depuration (kd ± standard error), half-life (t1/2 ± standard error), kinetically
derived bioconcentration factor (BCFk ± standard error, estimated by ku/kd) and steady-state bioconcentration factor (BCFss, estimated
by Cf /Cw). Regression coefficients (r2) are shown in parentheses.
BCFk BCFss
-1 -1
(tissues, L×kg ) (tissues, L×kg )
-1 -1
Test Compound ku (L×kg ×d ) -1
kd (d ) Half-life (t1/2, d) (plasma, dimensionless) (plasma, dimensionless)
Plasma-Low
Naproxen 108 ± 246 (0.35) 2.44 ± 0.28 (1.00) 0.28 ± 0.03 44.4 ± 101 4.36 ± 0.68
Ibuprofen 283 ± 758 (0.71) 3.82 ± 0.66 (1.00) 0.18 ± 0.03 74.2 ± 199 12.6 ± 4.65
Diclofenac 20.6 ± 46.3 (0.17) 1.75 ± 1.23 (0.68) 0.40 ± 0.28 11.8 ± 27.8 2.82 ± 0.94
Gemfibrozil 117 ± 121 (0.73) 3.62 ± 0.76 (1.00) 0.19 ± 0.04 32.4 ± 34.0 7.30 ± 1.48
Risperidone 9.36 ± 5.79 (0.63) 2.88 ± 0.21 (1.00) 0.24 ± 0.02 3.25 ± 2.03 3.74 ± 1.49
Diphenhydramine 6.35 ± 4.73 (0.72) 2.76 ± 0.88 (0.99) 0.25 ± 0.08 2.30 ± 1.87 0.59 ± 0.16
Fluoxetine 0.29 ± 0.05 (0.99) 1.90 ± 0.21 (0.99) 0.36 ± 0.04 0.15 ± 0.03 4.83 ± 0.29
Sertraline 1.11 ± 0.30 (0.87) 5.15 ± 0.70 (1.00) 0.13 ± 0.02 0.22 ± 0.07 3.95 ± 0.40
Carbamazepine 10.4 ± 16.8 (0.24) 2.50 ± 1.40 (0.93) 0.28 ± 0.15 4.16 ± 7.10 0.83 ± 0.26
Bisphenol A ND ND ND ND ND
Simvastatin ND ND ND ND ND
Triclosan 7.58 ± 5.60 (0.38) 1.20 ± 0.57 (0.83) 0.58 ± 0.27 6.31 ± 5.55 51.3 ± 12.4
Norfluoxetine b 2.68 ± 0.18 (0.93) 0.29 ± 0.07 (0.89) 2.43 ± 0.64 9.41 ± 2.54 8.54 ± 3.55
Plasma-High
Naproxen 187 ± 315 (0.31) 2.81 ± 0.33 (1.00) 0.25 ± 0.03 66.7 ± 112 8.95 ± 0.72
Ibuprofen 357 ± 127 (0.84) 3.76 ± 0.56 (1.00) 0.18 ± 0.03 94.9± 338 11.2 ± 1.83
Diclofenac 14.2± 4.46 (0.87) 3.58 ± 0.53 (1.00) 0.19 ± 0.03 3.96 ± 1.38 2.52 ± 0.39
Gemfibrozil 214 ± 156 (0.82) 3.40 ± 0.16 (1.00) 0.20 ± 0.01 62.9 ± 46.1 14.6 ± 1.75
Risperidone 8.06 ± 1.97 (0.92) 2.34 ± 0.11 (1.00) 0.30 ± 0.01 3.44 ± 0.86 3.99 ± 1.03
Diphenhydramine 5.27 ± 1.54 (0.88) 2.22 ± 0.20 (1.00) 0.31 ± 0.03 2.37 ± 0.73 1.06 ± 0.20
Fluoxetine 2.22 ± 0.95 (0.69) 2.15 ± 0.06 (1.00) 0.32 ± 0.01 1.03 ± 0.44 4.26 ± 0.41
Sertraline 20.9 ± 5.45 (0.91) 2.96 ± 0.01 (1.00) 0.23 ± 0.00 7.06 ± 1.84 3.67 ± 0.85
Carbamazepine 22.2 ± 25.3 (0.51) 2.44 ± 0.60 (0.99) 0.28 ± 0.07 9.13 ± 10.6 1.45 ± 0.14
Bisphenol A 1.20 ± 0.78 (0.39) 2.05 ± 0.08 (1.00) 0.34 ± 0.01 0.58 ± 0.38 5.32 ± 2.57

S9
 Simvastatin ND ND ND ND ND
Triclosan 7.98 ± 2.54 (0.87) 0.45 ± 0.17 (0.84) 1.53 ± 0.56 17.6 ± 8.52 46.6 ± 14.12
Norfluoxetine b 2.49 ± 0.12 (0.96) 0.27 ± 0.07 (0.86) 2.54 ± 0.65 9.13 ± 2.40 7.65 ± 2.07
Liver-Low
Naproxen 70.2 ± 88.0 (0.55) 2.74 ± 1.17 (0.97) 0.25 ± 0.11 25.7 ± 34.0 3.82 ± 0.98
Ibuprofen FF 3.54 ± 0.95 (1.00) 0.20 ± 0.05 NA 2.69 ± 0.35
Diclofenac FF 3.83 ± 0.64 (1.00) 0.18 ± 0.03 NA 1.11 ± 0.41
Gemfibrozil 9.91 ± 4.19 (0.62) 2.74 ± 0.86 (0.99) 0.25 ± 0.08 3.62 ± 1.91 22.5 ± 2.17
Risperidone 52.5 ± 21.9 (0.78) 1.91 ± 0.71 (0.94) 0.36 ± 0.13 27.4 ± 15.3 15.9 ± 5.59
Diphenhydramine FF 2.55 ± 0.32 (1.00) 0.27 ± 0.03 NA 3.38 ± 1.71
Fluoxetine 79.1 ± 30.6 (0.80) 0.90 ± 0.26 (0.91) 0.77 ± 0.22 87.9 ± 42.2 41.6 ± 2.52
Sertraline 333 ± 129 (0.83) 1.59 ± 0.40 (0.96) 0.44 ± 0.11 209 ± 96.7 64.7 ± 10.7
Carbamazepine 2.33 ± 2.28 (0.46) 0.29 ± 0.07 (0.90) 2.38 ± 0.58 7.99 ± 8.05 1.24 ± 0.63
Bisphenol A 46 ± 14 (0.90) 1.33 ± 0.39 (0.93) 0.52 ± 0.15 34.6 ± 14.7 183 ± 134
Simvastatin FF FF NA NA 57.1 ± 47.3
Triclosan 3850 ± 725 (0.94) 0.56 ± 0.19 (0.86) 1.24 ± 0.42 6870 ± 2650 6460 ± 1560
Norfluoxetine b 36.4 ± 4.52 (0.64) 0.17 ± 0.09 (0.57) 4.09 ± 2.22 215 ± 119 112 ± 43.4
Liver-High
Naproxen 166 ± 348 (0.47) 3.11 ± 0.45 (1.00) 0.22 ± 0.03 53.4 ± 112 6.12 ± 4.26
Ibuprofen FF 3.70 ± 0.30 (1.00) 0.19 ± 0.02 NA 2.26 ± 0.37
Diclofenac 0.19 ± 0.05 (0.90) 3.66 ± 1.77 (0.99) 0.19 ± 0.09 0.05 ± 0.03 0.51 ± 0.07
Gemfibrozil 113 ± 109 (0.37) 2.20 ± 0.25 (1.00) 0.31 ± 0.04 51.3 ± 49.7 19.1 ± 3.99
Risperidone 36.7 ± 10.1 (0.90) 2.18 ± 0.23 (1.00) 0.32 ± 0.03 16.8 ± 4.97 14.0 ± 3.86
Diphenhydramine 21.2 ± 9.90 (0.73) 2.80 ± 0.12 (1.00) 0.25 ± 0.01 7.56 ± 3.55 4.67 ± 1.05
Fluoxetine 83.3 ± 28.19 (0.84) 1.29 ± 0.18 (0.98) 0.54 ± 0.07 64.8 ± 23.6 43.0 ± 4.11
Sertraline 522 ± 83.53 (0.97) 1.73 ± 0.22 (0.99) 0.40 ± 0.05 302 ± 61.3 79.4 ± 11.8
Carbamazepine 9.19 ± 6.19 (0.58) 3.92 ± 3.67 (0.98) 0.18 ± 0.17 2.35 ± 2.70 1.85 ± 0.71
Bisphenol A 99.5 ± 37.51 (0.79) 0.78 ± 0.06 (0.99) 0.89 ± 0.07 128 ± 49.4 183 ± 91.1
Simvastatin 8.26 ± 4.06 (0.94) 1.89 ± 1.04 (0.85) 0.37 ± 0.20 4.37 ± 3.24 151± 136
Triclosan 8610 ± 1920 (0.94) 0.65 ± 0.03 (1.00) 1.07 ± 0.05 13300 ± 3040 4730 ± 1500
Norfluoxetine b 35.1 ± 5.15 (0.56) 0.20 ± 0.05 (0.88) 3.52 ± 0.83 179 ± 49.6 99.6 ± 13.17

S10
 Muscle-Low
Naproxen 8.20 ± 6.51 (0.65) 2.58 ± 1.02 (0.97) 0.27 ± 0.11 3.18 ± 2.81 0.73 ± 0.11
Ibuprofen 5.37 ± 3.99 (0.64) 0.32 ± 0.14 (0.62) 2.20 ± 0.96 17.1 ± 14.7 0.53 ± 0.08
Diclofenac FF FF NA NA 0.18 ± 0.09
Gemfibrozil 16.9 ± 12.3 (0.78) 0.70 ± 0.21 (0.87) 0.99 ± 0.29 24.1 ± 18.9 1.43 ± 0.38
Risperidone 2.28 ± 0.37 (0.95) 0.52 ± 0.12 (0.91) 1.34 ± 0.31 4.42 ± 1.25 6.50 ± 2.10
Diphenhydramine 2.30 ± 1.32 (0.74) 1.59 ± 0.10 (1.00) 0.44 ± 0.03 1.44 ± 0.83 1.12 ± 0.27
Fluoxetine 5.40 ± 2.52 (0.73) 1.02 ± 0.15 (0.98) 0.68 ± 0.10 5.29 ± 2.58 2.25 ± 0.40
Sertraline 40.9 ± 18.6 (0.81) 1.59 ± 0.18 (0.99) 0.44 ± 0.05 25.8 ± 12.1 5.32 ± 1.10
Carbamazepine 23.1 ± 32.2 (0.83) FF NA NA 1.21 ± 0.21
Bisphenol A 16.4 ± 14.3 (0.64) 0.19 ± 0.13 (0.41) 3.57 ± 2.36 84.4 ± 92.2 1.57 ± 1.13
Simvastatin FF FF NA NA 329 ± 116 a
Triclosan 946 ± 252 (0.92) 0.18 ± 0.06 (0.75) 3.83 ± 1.23 5220 ± 2170 136 ± 36.7
Norfluoxetine b 1.90 ± 0.31 (0.73) 0.49 ± 0.07 (0.96) 1.42 ± 0.21 3.89 ± 0.87 5.14 ± 2.61
Muscle-High
Naproxen 13.2 ± 10.8 (0.56) 2.27 ± 0.21 (1.00) 0.31 ± 0.03 5.80 ± 4.79 1.12 ± 0.13
Ibuprofen 4.67 ± 1.33 (0.91) 2.10 ± 0.47 (0.98) 0.33 ± 0.07 2.22 ± 0.81 0.65 ± 0.11
Diclofenac 0.25 ± 0.15 (0.66) 0.52 ± 0.05 (0.99) 1.33 ± 0.13 0.47 ± 0.29 0.09 ± 0.03
Gemfibrozil 28.1 ± 14.2 (0.85) 2.78 ± 0.77 (0.99) 0.25 ± 0.07 10.1 ± 5.81 2.62 ± 0.47
Risperidone 1.70 ± 0.38 (0.91) 0.52 ± 0.13 (0.89) 1.33 ± 0.34 3.27 ± 1.11 5.01 ± 0.92
Diphenhydramine 4.96 ± 0.81 (0.96) 1.43 ± 0.07 (1.00) 0.49 ± 0.02 3.47 ± 0.59 1.12 ± 0.10
Fluoxetine 4.51 ± 1.65 (0.82) 1.06 ± 0.06 (1.00) 0.65 ± 0.04 4.25 ± 1.57 2.10 ± 0.23
Sertraline 57.5 ± 21.9 (0.85) 1.59 ± 0.14 (1.00) 0.44 ± 0.04 36.2 ± 14.1 8.72 ± 1.81
Carbamazepine 3.68 ± 1.98 (0.74) FF NA NA 0.43 ± 0.10
Bisphenol A 1.13 ± 0.52 (0.58) 0.92 ± 0.14 (0.97) 0.75 ± 0.11 1.22 ± 0.59 2.43 ± 1.24
Simvastatin FF FF NA NA 143 ± 124
Triclosan 87.4 ± 21.2 (0.88) 0.73 ± 0.14 (0.95) 0.95 ± 0.18 120 ± 36.5 122 ± 41.3
Norfluoxetine b 1.82 ± 0.17 (0.89) 0.42 ± 0.17 (0.74) 1.66 ± 0.66 4.35 ± 1.79 4.28 ± 0.59
Ovary-Low
Naproxen FF 2.83 ± 0.70 (0.99) 0.24 ± 0.06 NA 0.42 ± 0.09
Ibuprofen FF FF NA NA 0.90 ± 0.32

S11
 Diclofenac FF FF NA NA NA
Gemfibrozil FF 1.88 ± 2.23 (0.67) 0.37 ± 0.44 NA 0.20 ± 0.05
Risperidone 0.19 ± 0.07 (0.71) 1.90 ± 0.06 (1.00) 0.36 ± 0.01 0.10 ± 0.04 53.1 ± 21.6
Diphenhydramine 3.02 ± 1.72 (0.40) 1.26 ± 0.08 (1.00) 0.55 ± 0.04 2.40 ± 1.37 13.8 ± 5.61
Fluoxetine 1.14 ± 0.08 (0.99) 0.38 ± 0.01 (1.00) 1.81 ± 0.06 2.99 ± 0.24 48.4 ± 2.93
Sertraline 0.88 ± 0.20 (0.92) 0.51 ± 0.01 (1.00) 1.35 ± 0.04 1.71 ± 0.40 126 ± 12.6
Carbamazepine 2.93 ± 1.17 (0.31) 3.24 ± 1.77 (0.98) 0.21 ± 0.12 0.90 ± 0.61 2.55 ± 0.65
Bisphenol A 1.87 ± 0.66 (0.75) 1.22 ± 0.19 (0.98) 0.57 ± 0.09 1.54 ± 0.59 12.1 ± 8.65
Simvastatin FF 0.14 ± 0.09 (0.24) 5.06 ± 3.36 NA 897 ± 164 a
Triclosan 0.56 ± 0.48 (0.44) 5.14 ± 65.5 (0.68) 0.13 ± 1.72 0.11 ± 1.39 226 ± 127
Norfluoxetine b 38.4 ± 3.22 (0.94) 0.09 ± 0.04 (0.57) 7.43 ± 3.49 412 ± 196 200 ± 80.4
Ovary-High
Naproxen FF 2.73 ± 0.39 (1.00) 0.25 ± 0.04 NA 1.06 ± 0.05
Ibuprofen FF 3.53 ± 1.11 (1.00) 0.20 ± 0.06 NA 1.43 ± 0.48
Diclofenac 0.25 ± 0.15 (0.66) 0.41 ± 0.14 (0.86) 1.67 ± 0.57 0.59 ± 0.41 0.17 ± 0.11
Gemfibrozil FF 2.74 ± 1.20 (0.97) 0.25 ± 0.11 NA 1.35 ± 0.45
Risperidone 1.78 ± 0.42 (0.85) 1.73 ± 0.09 (1.00) 0.40 ± 0.02 1.03 ± 0.25 28.2 ± 6.78
Diphenhydramine 14.0 ± 3.69 (0.80) 0.96 ± 0.12 (0.98) 0.73 ± 0.09 14.6 ± 4.31 12.4 ± 1.09
Fluoxetine 7.17 ± 0.46 (0.99) 0.33 ± 0.05 (0.96) 2.09 ± 0.34 21.7 ± 3.73 47.2 ± 4.51
Sertraline 5.94 ± 0.74 (0.97) 0.39 ± 0.09 (0.93) 1.77 ± 0.39 15.2 ± 3.88 171 ± 25.3
Carbamazepine 7.30 ± 2.98 (0.56) 2.12 ± 0.24 (1.00) 0.33 ± 0.04 3.45 ± 1.46 3.00 ± 0.30
Bisphenol A 7.03 ± 2.45 (0.68) 1.18 ± 0.10 (0.99) 0.59 ± 0.05 5.94 ± 2.13 21.9 ± 10.5
Simvastatin FF FF NA NA 641 ± 544
Triclosan FF 1.07 ± 0.87 (0.66) 0.65 ± 0.52 NA 104 ± 37.6
Norfluoxetine b 40.8 ± 3.09 (0.95) 0.12 ± 0.02 (0.90) 5.91 ± 1.15 347 ± 72.7 196 ± 18.5
∑Tissues-Low
Naproxen 3.14 ± 0.56 (0.86) 2.65 ± 0.80 (0.98) 0.26 ± 0.08 1.18 ± 0.30 0.80 ± 4.11
Ibuprofen 1.82 ± 0.44 (0.56) 0.66 ± 0.25 (0.80) 1.05 ± 0.40 2.76 ± 0.16 0.88 ± 9.11
Diclofenac FF FF NA NA 0.18 ± 7.46
Gemfibrozil 2.82 ± 0.43 (0.52) 1.29 ± 0.16 (0.99) 0.54 ± 0.07 2.18 ± 0.09 2.05 ± 3.22
Risperidone 33.7 ± 3.35 (0.75) 1.60 ± 0.11 (1.00) 0.43 ± 0.03 21.09 ± 0.01 23.4 ± 5.21

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 Diphenhydramine 7.21 ± 0.89 (0.42) 1.31 ± 0.07 (1.00) 0.53 ± 0.03 5.51 ± 0.02 5.69 ± 1.96
Fluoxetine 8.06 ± 0.62 (0.91) 0.42 ± 0.01 (1.00) 1.64 ± 0.05 19.1 ± 0.00 20.4 ± 1.24
Sertraline 26.0 ± 2.70 (0.81) 0.57 ± 0.03 (1.00) 1.22 ± 0.05 45.6 ± 0.00 50.9 ± 5.15
Carbamazepine FF FF NA NA 1.68 ± 5.14
Bisphenol A 12.5 ± 1.83 (0.69) 1.18 ± 0.23 (0.97) 0.59 ± 0.12 10.6 ± 0.02 13.8 ± 9.91
Simvastatin FF FF NA NA 514 ± 38.0 a
Triclosan 303 ± 17.8 (0.91) 0.59 ± 0.16 (0.90) 1.18 ± 0.31 517 ± 0.00 465 ± 113
Norfluoxetine b 12.9 ± 0.34 (0.99) 0.10 ± 0.04 (0.62) 6.75 ± 2.92 125 ± 54.2 79.1 ± 31.9
∑Tissues-High
Naproxen 6.05 ± 1.11 (0.51) 2.54 ± 0.24 (1.00) 0.27 ± 0.03 2.39 ± 0.09 1.41 ± 8.11
Ibuprofen 4.13 ± 0.61 (0.35) 2.98 ± 0.30 (1.00) 0.23 ± 0.02 1.39 ± 0.13 1.11 ± 4.17
Diclofenac FF FF NA NA 0.16 ± 2.23
Gemfibrozil 10.1 ± 1.39 (0.55) 2.59 ± 0.54 (0.99) 0.27 ± 0.06 3.90 ± 0.06 3.07 ± 9.37
Risperidone 18.1 ± 1.04 (0.84) 1.33 ± 0.19 (0.98) 0.52 ± 0.08 13.6 ± 0.01 13.6 ± 2.58
Diphenhydramine 5.92 ± 0.52 (0.56) 1.03 ± 0.10 (0.99) 0.68 ± 0.07 5.77 ± 0.02 5.25 ± 9.46
Fluoxetine 8.25 ± 0.24 (0.99) 0.37 ± 0.03 (0.99) 1.85 ± 0.17 22.0± 0.00 20.0 ± 1.91
Sertraline 31.4 ± 1.86 (0.92) 0.43 ± 0.07 (0.96) 1.62 ± 0.27 73.4 ± 0.00 69.4 ± 17.3
Carbamazepine 2.54 ± 0.33 (0.35) 1.43 ± 0.55 (0.88) 0.48 ± 0.19 1.78 ± 0.23 1.41 ± 7.13
Bisphenol A 14.2 ± 1.55 (0.75) 0.92 ± 0.08 (0.99) 0.75 ± 0.06 15.3 ± 0.01 17.9 ± 8.62
Simvastatin FF FF NA NA 318± 256
Triclosan 265 ± 26.5 (0.64) 0.68 ± 0.05 (0.99) 1.02 ± 0.07 388.6 ± 0.00 332 ± 191
b
Norfluoxetine 16.7 ± 0.93 (0.97) 0.12 ± 0.02 (0.93) 5.59 ± 0.90 135± 22.9 76.1 ± 7.30
a
Calculated by concentration at steady state divided by MDL (0.010 ng/mL), as the chemical concentration in water is lower than
MDL
< 0.05), hence BCFss values may potentially be higher than the reported value, which was calculated as the observed plasma or tissue
concentration on day 6 divided by day 6 water concentration.
b
These are pseudo-ku and pseudo-BCFss values, as norfluoxetine was not present in the dosed water, hence values were estimated
based on the observed concentrations of fluoxetine in the exposure tank water.
FF: Fitting failed
NA: Not available
ND: Not detected in tissue

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Table S7. Observed uptake and elimination rate constants (ku and kd) and steady-state bioconcentration factor (BCFss) of fluoxetine, as
well as those of pseudo-BCFss of norfluoxetine and total fluoxetine (fluoxetine + norfluoxetine) in ∑Tissues.

ku kd Half-life BCFss
-1
(L×kg ×d )
-1 -1
(d ) (t1/2, d) (L×kg-1)
Low Exposure Group
Fluoxetine 8.06 ± 0.62 0.42 ± 0.01 1.64±0.05 20.4 ± 1.24
Norfluoxetine 12.9 ± 0.34 0.10 ± 0.04 (0.62) 6.75 ± 2.92 79.1 ± 31.0

Total Fluoxetine (Fluoxetine + 21.8 ± 1.22 0.13 ± 0.04 (0.80) 5.31 ± 1.57 99.5 ± 41.0
Norfluoxetine)
- - - 79%
% Reduction in BCFss due to
biotransformation

High Exposure Group

Fluoxetine 8.25 ± 0.24 0.37 ± 0.03 1.85±0.17 20.0 ± 1.91

Norfluoxetine 16.7 ± 0.93 0.12 ± 0.02 (0.93) 5.59 ± 0.90 76.1 ± 7.30

Total Fluoxetine (Fluoxetine + 23.3 ± 0.89 0.15 ± 0.02 (0.97) 4.52 ± 0.50 96.1 ± 10.14
Norfluoxetine)

% Reduction in BCFss due to - - - 79%

biotransformation

S14
 Table S8. Comparison of depuration rate constants (kd ± standard error), and steady-state bioconcentration factor (BCFss) for the
various of the studied PPCPs and various perfluoroalkyl substances.

kd (d-1) in liver BCFss (L×kg-1) in liver

Log Dmw
Chemical Low Group High Group Low Group High Group
(pH=7) a
Naproxen 1.46 2.74 ± 1.17 (0.97) 3.11 ± 0.45 (1.00) 3.82 ± 0.98 6.12 ± 4.26
PFHxS 2.50 0.151 ± 0.006 (0.59) 0.068 ± 0.002 (0.94) 365 ± 75.8 247 ± 54.5
Ibuprofen 1.81 3.54 ± 0.95 (1.00) 3.70 ± 0.30 (1.00) 2.69 ± 0.35 2.26 ± 0.37
Diclofenac 2.66 3.83 ± 0.64 (1.00) 3.66 ± 1.77 (0.99) 1.11 ± 0.41 0.51 ± 0.07
PFOS 2.65 0.023 ± 0.01 (0.26) 0.180 ± 0.003 (0.96) 4410 ± 1220 3560 ± 1190
Gemfibrozil 3.11 2.74 ± 0.86 (0.99) 2.20 ± 0.25 (1.00) 22.5 ± 2.17 19.1 ± 3.99
Risperidone 2.78 1.91 ± 0.71 (0.94) 2.18 ± 0.23 (1.00) 15.9 ± 5.59 14.0 ± 3.86
Diphenhydramine 2.74 2.55 ± 0.32 (1.00) 2.80 ± 0.12 (1.00) 3.38 ± 1.71 4.67 ± 1.05
Fluoxetine 3.71 0.90 ± 0.26 (0.91) 1.29 ± 0.18 (0.98) 41.6 ± 2.52 43.0 ± 4.11
PFOA 2.98 0.107 ± 0.032 (0.89) 0.185 ± 0.026 (0.99) 247 ± 65.4 266 ± 48.4
Sertraline 4.33 1.59 ± 0.40 (0.96) 1.73 ± 0.22 (0.99) 64.7 ± 10.7 79.4 ± 11.8
Carbamazepine 3.80 0.29 ± 0.07 (0.90) 3.92 ± 3.67 (0.98) 1.24 ± 0.63 1.85 ± 0.71
PFNA 3.65 0.036 ± 0.010 (0.80) 0.093 ± 0.018 (0.94) 1530± 421 2790 ± 648
Bisphenol A 4.41 1.33 ± 0.39 (0.93) 0.78 ± 0.06 (0.99) 183 ± 134 183 ± 91.1
PFDPA 4.16 0.022 ± 0.017 (0.17) 0.095 ± 0.033 (0.81) 1430 ± 737 772 ± 248
b
Simvastatin 4.85 NA 1.89 ± 1.04 (0.85) 57.1 ± 47.3 151 ± 136
4
PFDA 4.33 NA 0.014 ± 0.002 (0.94) 1.1×10 ± 1340 2.3×104 ± 4410
Triclosan 5.47 0.56 ± 0.19 (0.86) 0.65 ± 0.03 (1.00) 6460 ± 1560 4730 ± 1500
5 4
C6/C6 PFPiA 5.15 0.036± 0.014 (0.66) NA 3.2×10 ± 9.9×10 3.7×104 ± 1.4×104
PFUnDA 5.01 NA 0.015 ± 0.012 (0.20) 1.4×104 ± 4.2 ×103 1.4×104 ± 3.6×103
a
Dmw (pH 7, L/kg) values were estimated following the approach outlined by Armitage et al..8 Specifically, the liposome-water partition coefficient for neutral species was
determined as Log Kmw, N = 1.01 × Log Kow + 0.12, the liposome-water partition coefficient for ionic species was determined as Log Kmw, I = Log Kmw,N - Dmw. Subsequently,
Dmw was determined as, Dmw (pH 7) = fN × Kmw, N + fI × Kmw, I where fN is the fraction of chemical in neutral form and fI is the fraction of chemical in charged form at pH 7, as
predicted by the Henderson-Hasselbalch equation. bNot available

S15
Figure S1. Growth-corrected uptake and depuration phase concentrations (n=3, mean ± SD) for PPCPs in plasma (A), liver (B), ovary
(C) and muscle (D).

S16
 S17
 S18
 S19
Figure S2. Observed steady-state bioconcentration factor (log BCFss) versus kinetically derived bioconcentration factor (i.e., log BCFk)
predicted from the first-order kinetic model (i.e., ku/kd) in different tissues. The solid lines represent perfect model agreement 1:1.
Dashed lines represent 1 log unit interval of predicted concentrations. X axis error bar represents the standard error of predicted log
BCF and y axis error bar represents the standard deviation of observed log BCF.

S20
Figure S3. Relationship between steady-state bioconcentration factors (log BCFss, L×kg-1) of individual test chemicals in åtissues
versus physical-chemical properties, including log Dow, log Dmw, log Dmpw and log DBSAw. Plotted data are for both low and high
exposure groups. Data for risperidone are not plotted due to lack of available log DBSAw and log Dmpw values.

S21
Figure S4. Relationship between uptake rate constant (ku, L×kg-1×d-1) and depuration rate constant (kd, d-1) for ∑tissues versus
physical-chemical properties, including log Dow, log Dmw, log Dmpw and log DBSAw. Plotted data are observations for both low and high
exposure experimental groups. Data for risperidone are not plotted due to lack of available log DBSAw and log Dmpw values.

S22
Figure S5. Growth-corrected uptake and depuration phase concentrations (ng×g-1, n=3, mean ± SD) for fluoxetine and norfluoxetine in
liver, plasma, ovary and muscle.

S23
Supplemental References

(1) Martin, J. W.; Mabury, S. A.; Solomon, K. R.; Muir, D. C. Bioconcentration and tissue
distribution of perfluorinated acids in rainbow trout (Oncorhynchus mykiss). Environ.
Toxicol. Chem. 2003, 22 (1), 196-204.
(2) Bruggeman, W.; Martron, L.; Kooiman, D.; Hutzinger, O. Accumulation and elimination
kinetics of di-, tri-and tetra chlorobiphenyls by goldfish after dietary and aqueous exposure.
Chemosphere 1981, 10 (8), 811-832.
(3) Grandjean, P.; Budtz-Jørgensen, E. Immunotoxicity of perfluorinated alkylates: calculation
of benchmark doses based on serum concentrations in children. Environ. Health 2013, 12
(1), 35.
(4) Calafat, A. M.; Kuklenyik, Z.; Reidy, J. A.; Caudill, S. P.; Tully, J. S.; Needham, L. L.
Serum concentrations of 11 polyfluoroalkyl compounds in the U.S. population: Data from
the national health and nutrition examination survey (NHANES) 1999-2000. Environ. Sci.
Technol. 2007, 41 (7), 2237-2242.
(5) Draft toxicological profile for perfluoroalkyls. Agency for Toxic Substances and Disease
Registry: Atlanta, GA, 2009.
(6) Lindstrom, A. B.; Strynar, M. J.; Libelo, E. L. Polyfluorinated compounds: past, present,
and future. Environ. Sci. Technol. 2011, 45 (19), 7954-7961.
(7) Henneberger, L.; Goss, K.-U.; Endo, S. Partitioning of Organic Ions to Muscle Protein:
Experimental Data, Modeling, and Implications for in Vivo Distribution of Organic Ions.
Environ. Sci. Technol. 2016, 50 (13), 7029-7036.
(8) Armitage, J. M.; Arnot, J. A.; Wania, F.; Mackay, D. Development and evaluation of a
mechanistic bioconcentration model for ionogenic organic chemicals in fish. Environ.
Toxicol. Chem. 2013, 32 (1), 115-128.

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